This application is a 371 of PCT/AU95/00677 filed Oct. 13, 1995.
The present invention relates to new polyunsaturated fatty acids having antimalarial activity and/or neutrophil stimulatory activity. The present invention further relates to a group of modified polyunsaturated fatty acids which have the ability to suppress cytokine production and cytokine action. Such fatty acids have enhanced stability when compared to naturally occurring polyunsaturated fatty acids. The present invention further relates to compositions including the polyunsaturated fatty acids as the active ingredient and methods of anti-malarial, anti-infective of anti-inflammatory treatment or prevention involving the administration of this composition.
Over half of the world""s population is at risk from malaria, with about 500 million acute infections and approximately 1 million deaths recorded each year. (Tropical Diseases Progress in International Research, 1987-1988. Ninth Programme Report, UNDP/World Bank/WHO, Geneva, 43-49: Stevenson MM Preface In: Stevenson MM. Ed. Malaria: Host responses to Infection, CRC Press, Inc). The use of antimalarial drugs is associated with major problems because of increased resistance and toxic side-effects. Most currently used antimalarials are unsuitable for use in children (most at risk of potentially fatal cerebral malaria), pregnant women and the aged.
Inflammation may be caused by bacteria, viruses and/or other infective agents, opportunistic infections (which may be consequent on an immunodepressed state, for example resulting from cancer or therapy, particularly cytotoxic drug therapy or radiotherapy), autoimmunity of otherwise. Septic shock is an illustration of a disease involving inflammation. Many of the clinical features of Gram-negative septic shock may be reproduced in animals by the administration of LPS to animals can prompt severe metabolic and physiological changes which can lead to death. Associated with the injection of LPS is the extensive production of pro-inflammatory cytokines such as tumour necrosis factor alpha [TNFxcex1]. Cachexia, which is characteristic of chronic exposure to TNF or interleukin-6, is a common symptom of advanced malignancy and severe infection. It is characterised by abnormal protein and glucose metabolism and body wasting. Chronic administration of TNF IL-1 in mice, rats and/or humans cause anorexia, weight loss and depletion of body lipid and protein within 7 to 10 days (Cerami et al. 1985, Immunol. Lett. 11, 173: Fong et al, 1989J. Exp. Med. 170, 1627, Moldawer et al. Am. J. Phusiol., 254 G450-G456, 1988: Fong et am. Am. J Physiol, 256, R659-R665 (1989): McCarthy et al. Am. J. Clin. Nature, 42, 1179-1182, 1982). TNF levels have been measured in patients with cancer and chronic disease associated with cachexia.
TNFxcex1 and IL-1, with their common functional activities such as pyrogenicity, somnogenicity and being mediators of inflammation, have been implicated in the pathology of other diseases associated with chronic inflammation apart from toxic shock and cancer-related cachexia. TNF has been detected in synovial fluid in patients with both rheumatoid and reactive arthritis and in the serum of patients with rheumatoid arthritis (Saxne et en. 1988, Arthrit. Rheumat. 31, 1041). Raised levels of TNF have been detected in renal transplant patients during acute rejection episodes (Maury and Teppo 1987, J. Exp. Med. 166, 1132). In animals, TNF has been shown to be involved in the pathogenesis of graft-versus-host disease in skin and gut following allogenic marrow transplantation.
Administration of a rabbit anti-murine TNF antibody was shown to prevent the histological changes associated with graft-versus-host disease and to reduce mortality (Piquet et en. 1987, J. Exp. Med. 166, 1220). TNF has also been shown to contribute significantly to the pathology of malaria (Clark et al. 1987, Am. J. Pathol. 129, 192-199). Further, elevated serum lebvels of TNF have been reported in malaria patients (Scuderi et al. 1986, Lancet 2, 1364-1365).
Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system and is the commonest chronic neutroligical disease of young adults. The incidence of MS and its pattern of distribution have been unchanged for decades. The disease remains essentially untreatable.
MS usually affects multiple areas of white matter in the central nervous system (CNS), most frequently, the preventricual white matter, brainstem, spinal cord and the optic nerves. The primary process destroys myelin sheaths and eventually kills oligodendrocytes creating the characteristic plaque of MS.
The early development of the plaque is characterised by the development of perivascular inflammation followed by the migration of lymphocytes, plasma cells and macrophages into the lesion. This is followed by astrocyte gliosis and the attempts of demyelination by oligodendrocytes. The plaque is surrounded by lymphocytes. Anti-T cell agents such as anti-CD4 treatment are effective. Such agents inhibit the proliferation of T-cells.
Although the aetiology of MS is still unknown, the focus of research efforts that have led to plausible hypotheses have been those of immune dysregulation including autoimmunity and genetic predisposition, both of which may play a role in the actual development of disease. Both TNFxcex1 (lymphotoxin) and TNFxcex1 are thought to play a role in the pathophysiology.
Multiple immunological abnormalities are reproducibly found in patients in the acute stage of the disease. The synthesis of immunoglobulins, although normal in the periphery, is increased in the central nervous system and the antibodies produced have a characteristic banding pattern. The antigenic, specificity of these antibodies is not known and it is unclear whether they have a role to play in the progression of the disease.
Various stressors known to activate the immune system such as viral infection or surgery can also produce an exacerbation of MS. Other activators such as xcex3-interferon produce similar effects when administered. In addition, immunosuppressive anti-inflammatory therapy with corticosteroids for example, can produce modest remission or at least palliation for short periods of time.
Myelopathy, a disorder of the spinal cord, can have many different aetiologies, most of which are mediated by inflammation, including the following:
Neurosyphillis:
b12 or folate deficiency:
sarcoidosis:
transverse myelitis:
arachidonitis:
cervical spondylitis:
motor neuron disease:
neurofibromatosis:
spinal cord compression from tumour, disc or arthritis:
lupus erythematosus of the spinal cord: and
viral encephalomyelitis
Chronic inflammation or, as more commonly known, chronic immune system activation occurs in response to persistent antigen whose origin may be exogenous or may result from an autoimmune state. Such chronic inflammation results in local tissue destruction and depending upon the type of inflammation can result in systemic effects due to the sustained production of inflammatory mediators. Such inflammatory mediators include the cytokines which are soluble mediators produced by activated lymphocytes and macrophages and effect cellular communication and physiological response. Chronic immune activation can occur as a result of infectious disease, such as chronic fatigue syndrome or toxic shock syndrome or through autoimmune mechanisms resulting in such conditions as rheumatoid arthritis, inflammatory bowel disease, Crohns Disease and other diseases such as graft versus host disease.
Rheumatoid arthritis [Marrow et al. I xe2x80x9cAutoimmune Rheumatic Diseasexe2x80x9d, Blackwell Scientific Publ. Oxford, UK, Chapter 4 pp148-207 (1987)] is a disease characterised by chronic inflammation and erosion of joints that may affect up to 3% of the population, including children. Symptoms of rheumatoid arthritis include morning stiffness, swelling and pain upon motion in at lease one joint and joint swelling. Non-specific symptoms including lethargy, anorexia and weakness as well as fever and lymphadenopathy (characteristic of immune activation) may antedate joint involvement. Extra-articular manifestations of rheumatoid arthritis include vasculitis, cataracts, uveitis, interstitial fibrosis, pericarditis and myocarditis, peripheral neuropathy, myeloid deposits, chronic anaemia and subcutaneous and pulmonary nodules.
Genetic factors and infectious agents including bacteria, fungi, mycoplasmas and viruses have been associated with the development of rheumatoid arthritis. Mild rheumatoid arthritis may be treated with non-steroidal anti-inflammatory drugs while severe cases require systemic corticosteroids, anti-metabolites or cytotoxic agents. Experimentally, anti-CD4 monoclonal antibodies and anti-TNFxcex1 antibodies have been used to treat rheumatoid arthritis (Horneff et al, Cytokine 3 266-267 (1991): Horneff et al. Arth. Rheum. 34 129-140 (1991) and Shoenfeld et al, Clin. Exp. Rheum. 9, 663-673 (1991). Williams et al. 1992 PNAS 89, 9784).
Inflammatory bowel disease (IBD) and Crohns disease are chronic inflammatory conditions that fulfil some of the criteria of an autoimmune disease [Snook, Gut 31 961-963 (1991)]. Inflammation and tissue damage involves the recruitment and activation of neutrophils, macrophages and lymphocytes [MacDermott et al. Adv. Immunol. 42 285-328 (1988)] which generate cytokines and proinflammatory molecules such as prostaglandins and leukotrienes [MacDermott, Mt. Sinai J. Med. 57 273-278 (1990)]. As a result of chronic activation of immunocompetent cells, IL-1, IL-6[Starter, Immunol. Res. 10 273-278 (1990); Fiocchi, Immunol. Res. 10 239-248 (1991)] and TNFxcex1 [MacDermott, Mt. Sinai J. Med. 57 273-278 (1990)] are all elevated in IBD and Crohns Disease patients.
Drugs used to treat IBD and Crohns Disease include anti-inflammatory agents such as sulphasalazine (5-ASA) corticosteroids, cyclosporin A and azathiprine (Hanauer. Scand. J. Gastroenterol, 25 (Supl. 175) 97-106 (1990); Peppercorn. Annal, Intern, Med. 112 50-60 (1990)). Experimentally, anti-CD4 and anti-TNF monoclonal antibodies have been used to successfully treat ulcerative colitis (Emmerich et al. Lancet 338 570-571 (1991)).
Whilst a host may react against a genetically incompatible graft producing a host-versus-graft response, an immunocompetent graft (such as bone marrow or intestinal tissue) may react against the host resulting in graft-versus-host disease. These reactions are mediated by allogenic responses directed against a foreign MHC molecule and are mimicked in vitro by the mixed lymphocyte reaction (MLR). Graft/host interactions result in chronic inflammation surrounding the grafted tissue with an increase in markers of immune activation such as are seen in ADS (Grant. Immunol. Today 12 171-172 (1991)). Treatment of the graft/host interactions currently include either azathioprine, cyclosporin A or methylprednisone and, more recently, rapamycin (Spekowski et al. Transplantation 53 258‥264 (1992); Huber et al. Bibliotheca Cardiologica. 43 103-110 (1988)). Monoclonal antibodies specific for CD3 (Wissing et al. Clin Exp Immunol. 83 333-337 (1991)), CD4 (Reinke et al. Lancet 338 702-703 (1991)) and TNFxcex1 have been used experimentally to inhibit graft/host reactions.
as mentioned above PUFAs have a range of useful biological activities (see for example International Patent Application Nos. WO 93/00084 and WO 95/09622 and the references cited therein). Unfortunately, due to their limited stability in vivo. PUFAs have not achieved widespread use as therapeutic agents. The present inventors have developed substituted PUFAs which while retaining biological activity have increased stability in vivo i.e. slower metabolic turnover. These new polyunsaturated fatty acid (PUFA) compounds have direct antimalarial activity. In addition to their direct antimalarial activity, the novel PUFA activate human neutrophils causing release of granule contents, and exhibit synergy with TNF in the production of superoxide. Activation of human neutrophils by the PUFA results in enhanced ability of these cells to kill malaria parasite (P. falciparum) within red blood cells and also the bacteria Staphylococcus aureus. 
Further, the present inventors have also found that certain polyunsaturated fatty acids and novel polyunsaturated fatty acids and their hydroxy and hydroperoxy derivatives suppress production of cytokines.
These new PUFAs include at least one xcex2 oxa, xcex2 thia, xcex3 oxa or xcex3 thia substitution. While saturated xcex2-oxa fatty acids can be obtained using the standard procedure for either synthesis, by reaction of alkyl halides with the dianions of xcex1-hydroxy acids or by treating xcex1-halo acids with deprotonated alcohols, the unsaturated xcex2-oxa fatty acids of the present invention are not accessible using normal methods. Attempts to obtain the unsaturated compounds in this manner lead only to decomposition products, resulting from undesirable side reactions at the olefinic and allylic carbons.
In a recent variation of the standard procedure, saturated xcex2-oxa fatty acids have been obtained through nucleophilic substitution reactions under less vigorous conditions, by treating diazoacetates, activated by complexation with boron trifluoride etherate, with alcohols. However, boron trifluoride etherate is known to cause isomerization of alkenes and it is therefore unsuitable for use in the synthesis of unsaturated xcex2-oxa fatty acids.
The present inventors have now found that unsaturated xcex2-oxa fatty acids can be obtained, in good yields, by insertion of carbenes in the Oxe2x80x94H bond of alcohols. There is no complication from other carbene insertion reactions and, of particular significance, the olefinic moieties are unaffected under the reaction conditions.
The carbene can be generated from the corresponding diazo acetate or diazo alkane, by treatment with a catalyst such as a rhodium salt. Reaction of the carbene with the complementary alcohol, which is either a derivative of xcex1-hydroxy acetic acid or an unsaturated fatty alcohol affords the unsaturated xcex2-oxa fatty acid. In a preferred embodiment the alcohols are those obtained by reduction of naturally occurring unsaturated fatty acids or the corresponding esters, and reaction with an ester of diazo acetic acid affords the unsaturated xcex2-oxa fatty acid.
The present inventors have also shown that both xcex2-oxa and xcex2-thia substituted fatty acids are unable to undergo xcex2 oxidation. In addition certain of the novel compounds display other properties which differ from those of natural PUFA including enhanced solubility, varied oxidation reduction potentials and different charge and polarity.
Accordingly, in a first aspect the present invention consists in a polyunsaturated fatty acid compound having antimalarial and/or neutrophil stimulatory activity, the polyunsaturated fatty acid containing 18-25 carbons and 1-6 double bonds and wherein the polyunsaturated fatty acid compound has one or two substitutions selected from the group consisting of xcex2 oxa. xcex3 oxa, xcex2 thia and xcex3 thia.
In a preferred embodiment of the present invention the polyunsaturated fatty acid compound includes a further substitution selected from the group consisting of hydroxy, hydroperoxy, peroxy and carboxymethyl substitutions. In another embodiment the substituted fatty acid is covalently attached to an amino acid.
In a further preferred embodiment of the present invention the polyunsaturated fatty acid compound contains 20-25 carbon atoms and 3-6 double bonds and is preferably an n-3 to n-6 fatty acid.
In another preferred embodiment of the present invention the polyunsaturated fatty acid compound is 21 carbons with 3-4 double bonds containing a xcex2 oxa or xcex2 thia substitution, 22 carbon atoms with 3-4 double bonds containing a xcex3 thia or xcex2 oxa substitution. 23 carbons with 3-4 double bonds containing a xcex2 thia substitution. 24 carbons with 3-4 double bonds containing a xcex3 thia substitution. 25 carbons with 3-6 double bonds containing a xcex2 oxa substitution. 25 carbons with 3-6 double bonds containing a xcex2 thia substitution, or 23 carbons, 3-6 double bonds, xcex2 thia and xcex1-carboxymethyl group.
In yet another preferred embodiment of the present invention the polyunsaturated fatty acid compound has a xcfx89 hydroxy substitution.
In another preferred form of the invention the polyunsaturated fatty acid compound is attached to an amino acid, preferably an aspartic acid or glycine.
In a second aspect the present invention consists in a method of producing an unsaturated oxa substituted fatty acid comprising reacting an unsaturated fatty acid alcohol with a carbene such that the carbene is inserted in the Oxe2x80x94H bond of the alcohol.
In a preferred embodiment of this aspect of the present invention the unsaturated fatty acid alcohol contains 18-25 carbon atoms and 1-6 double bonds.
In a further preferred embodiment of this aspect of the present invention the carbene is synthesized via rhodium acetate catalysed of a diazo compound.
In another preferred embodiment of this aspect of the present invention an unsaturated xcex2 oxa substituted fatty acid is produced.
In a third aspect the present invention consists in a method of treating inflammation in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-inflammatory composition comprising at least one hydroxy, hydroperoxy, or peroxy derivative of a polyunsaturated fatty acid having a carbon chain of 18 to 24 carbon atoms and having 1-6 cis or trans double bonds.
In a preferred embodiment of this aspect of the present invention the poly unsaturated fatty acid may contain oxygen or sulphur atoms within the carbon chains as oxa or thia derivatives.
In a further preferred embodiment of this aspect of the present invention the polyunsaturated fatty acid is selected from the group consisting of the C20:4n-6 (5, 8, 11, 14-eicosatetraenoc acid). C20:5N-3 (5, 8, 11, 14, 17-eicosapentaenoic). C22:6n-3 (4, 7, 10, 13, 16, 19-docosahexaenoic acid) and arachidonic acid.
Neutrophil/macrophage stimulatory agents may have application in the treatment of other infections including Candida sp. Trypanosoma, Schistosomiasis, Tuberculosis, viruses eg herpes, Sindbis virus, Legionella, Listeriosis, Pneumocystsis, Pseudomonas. They would also be useful as adjunct therapy in immunocomporomised individuals including those undergoing cancer chemotherapy and transplant recipients and burns patients. In addition others, so called normal individuals may also be treated eg the aged, children under 2, alcoholics who are known to have poor phagocytic cell activity.